Homogeneous immune interferon fragment

ABSTRACT

An active fragment of human immune interferon and a method for the extraction and purification of this protein fragment is disclosed. This method permits the purification to homogenity of the active fragment of recombinant human immune interferon.

FIELD OF THE INVENTION

This invention relates to a homogeneous immune interferon fragment having full antiviral activity from a microorganism preparation which contains this protein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified recombinant human immune interferon in photo "A" reducing condition (0.7M-beta mercaptoethanol in the sample loading buffer) and in "B" non-reducing condition.

FIG. 2 illustrated the comparison between the predicted DNA sequence of recombinant human immune interferon and the actual DNA sequence of the 15K and 18K rIFN-γ species.

FIG. 3 illustrates the separation of C-terminal CNBr peptides of the 15K and 18K rIFN-γ.

BACKGROUND OF THE INVENTION

The prior art has devised various methods for effecting the extraction and purification of the family of anti-viral proteins known as interferon. To date there are three known major classes of interferon: IFN-α(leukocyte), INF-β(fibroblast) and IFN-γ(immune). Although the various interferon classes may be related in terms of anti-viral, anti-proliferative or structural arrangement, the prior art had so far been unable to devise a uniform method for the extraction and purification of all of these classes. Indeed, many of the processes useful for the extraction and purification of leukocyte interferon from a crude mixture of other proteins and cell debris would not work to extract and purify out fibroblast or immune interferon from the same kind of preparation.

The extraction step in the purification processes of the prior art typically involved either the mechanical (i.e. sonification) or chemical lysis of microorganisms which have produced, often through recombinant techniques, the desired foreign protein. During this mechanical or chemical lysis procedure various cellular proteases are also released into the mixture. These proteases are then free to enzymatically act upon and degrade the foreign protein in the mixture.

It has further been reported that certain amino acid sequences on immune interferon are not essential for antiviral activity. For instance, Capon et al. (reference 11) has reported that amino acids #136-146 of rIFN-γ are not essential for antiviral activity.

SUMMARY OF THE INVENTION

Broadly stated, this invention comprises a biologically active fragment of immune interferon isolated and purified from a preparation containing this protein. More specifically, this invention comprises a homogeneous 15K (15,000 dalton) immune interferon fragment species.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has now been discovered in the case of immune interferon, that extraction and purification with any of the conventional techniques described in the prior art will not yield an entirely homogeneous preparation of immune interferon with an intact of complete amino acid sequence. Only recently has recombinant technology advanced to the point at which it is possible to obtain sufficient quantitities of rINF-γ so as to characterize it and determine its amino acid sequence. When conventional prior art techniques were utilized to extract rINF-γ preparations and the amino acid sequence of purified material was determined, it was discovered that the purified preparation was, in fact, comprised of a variety of related protein species of different molecular weight. It has further now been discovered by amino acid sequencing that these proteins species are actually the intact sequence form of immune interferon in combination with a majority of proteolytic fragments of the intact sequence protein. Suprisingly, even though it has now been discovered that the prior art mixture contained both intact immune interferon protein and fragments thereof, it has been discovered that apparently a component of this mixture also retained biological activity.

The extraction of human rIFN-γ from transformed microorganisms producing and containing this protein can be carried out simply and efficiently with the use of sonification or mechanical lysis. However, it is believed that the rIFN-γ is degraded by proteases during the extraction step. Sonification of frozen cells yields a mixture of proteolytic products such as 15K rIFN-γ which results from the removal of C-terminal amino acid residues No. 132-146. The 15K r-IFN-γ after purification was found to be free of other proteolysic fragment and, significantly, had full biological activity equal to that of the complete 18K rIFN-γ protein (10⁷ units/mg) and comparable to that of homogeneous natural human immune interferon (reference 7). Amino acid compositions and N-terminal sequences of the 15K protein were consistent with that expected from the DNA sequence. The 15K rIFN-γ amino acid and corresponding DNA sequence is shown in FIG. 2 along with the amino acid and corresponding DNA sequence of the complete immune interferon protein. The DNA base sequence of the complete, 18K human rIFN-γ as used throughout this specification is also as described in reference 1.

Depending upon extraction procedures, different molecular weight (M.W.) species (about 15,000-18,000 daltons) of rIFN-γ have been separately purified. The purified interferon with apparent M.W. of 18,000 dalton on sodium dodecyl sulfate-polyacrylamide gel electrophoresis has been obtained by guanidine extraction. However, the lower 15,000 M.W. species immune interferon is preferably extracted by sonification in the absence of guanidine. The amino terminal sequence of both the 18K and 15K proteins were consistent with the sequence predicted from the DNA coding for the human immune interferon protein. It is also within the contemplation of this invention that the amino terminus of the immune interferon protein could begin with a Methonine (Met) as opposed to cystine (Cys) as shown in FIG. 2.

The C-terminal sequence of the 18K and 15K protein were determined by analyzing and sequencing purified C-terminal peptide after CNBr treatment. The amino acid composition and sequence of the C-terminal peptide released from the 18K rIFN-γ matched with the predicted sequence of rIFN-γ indicating that the 18K species is the intact molecule. On the other hand, a different CNBr cleaved peptide was released from the C-terminus of 15K rIFN-γ. Based on amino acid and sequence analysis, this different peptide corresponded to the amino acid residues No. 121-131 indicating that the 15K species was a proteolytic product.

In a preferred embodiment of this invention, the microorganisms employed as the recipient in the fermentation procedures and unless otherwise noted, is the microorganism Escherichia coli K-12 strain 294 as described in British Patent Publication No. 2055382A and which is incorporated by reference herein. This microorganism has been deposited with the American Type Culture Collection, ATCC Accession No. 31446, deposited Oct. 28, 1978. Furthermore, all recombinant DNA work herein was performed in compliance with applicable guidelines of the National Institutes of Health.

The invention, in its most preferred embodiments, is described with reference to E. coli, including not only E. coli K-12 strain 294, defined above, but also other known equivalent E. coli strains such as E. coli MA210 or RR1 (ATCC #31343), or other microbial strains many of which are publicly available or are deposited and available from recognized microorganism depository institutions, such as the American Type Culture Collection (ATCC)-cf. the ATCC catalog listing.

E. coli RR1 (pRK248cI_(ts), pRC231/IFN-γ) (See copending U.S. patent application Ser. No. 397,388 to Crowl filed July 12, 1982 for preparation of this microorganism, which application is incorporated by reference herein) was used in the following examples for rINF-γ fermentations. The pRK248cI_(ts) and pRC231/IFN-γ plasmids contained temperature sensitive repressor and IFN-γ gene respectively. Expression of rIFN-γ gene was under the control of P_(L) promoter.

Overnight cultures of E. coli RR1 (pRK248cI_(ts), pRC231/IF-γ) were grown in LB broth at 30° C. One liter of the overnight culture was diluted to 10 liters with minimal M-9 medium containing casamino acids. At logarithmic growth, the culture was shifted from 30° C. to 42° C. and continued to grow at 42° C. for 2-3 hours. Bacteria were harvested by centrifugation and the bacterial pellets were stored at -20° C. until used. All fermentations and procedures were performed in accordance with recombinant DNA guidelines of the National Institutes of Health.

Iodo [1-¹⁴ C] acetic acid was obtained from New England Nuclear, cyanogen bromide from Pierce Inc., carboxypeptidase A from Sigma Inc., fluorescamine from Hoffmann-La Roche Inc., C-8 and C-18 reverse-phase chromatographic columns from Supelco Inc. All the solvents used for protein characterization were re-distilled over ninhydrin.

Carboxymethylation of rIFN-γ by ¹⁴ C-iodoacetic acid was carried out in the presence of 6M guanidine HCl as described in published procedures of reference (3). Excess reagent was removed by HPLC on C8 reverse-phase column. Carboxypeptidase digestion was performed in 0.2M NH₄ HCO₃ as described in reference (4).

The rIFN-γ was treated with CNBr (100-fold molar excess over methionine) in 70 percent formic acid as described in reference (5), CNBr peptides were separated by HPLC on C-18 reverse-phase column. A linear gradient of 0 to 70 percent of CH₃ CN in 0.1% trifluoroacetic acid was used for peptide elution.

Protein or peptide samples were hydrolyzed for 20-24 hours in sealed, N₂ -flushed, evacuated tubes in constant boiling HCl containing 4% thioglyceric acid. Amino acid analyses were performed using a fluorescamine amino acid analyzer as described in reference (6).

An ABI (Applied Biosystems, Inc.) gas-phase sequencer 470A was used for sequence analyses of carboxymethylated proteins as described in reference (7). Samples of PTH-amino acids were identified by reverse-phase HPLC on an ultrasphere ODS column as described in reference (8).

As used herin the term "interferon activity" refers to the characteristic antiviral and antigrowth activity characteristic of the interferons. The characteristic antiviral activity of rIFN can be determined using the cytophatic effect inhibition test described in Familleti, P.C. et al., Methods in Enzymology 78, 387 (1981). The characteristic antigrowth activity of rIFN can be determined using the procedure described in Evinger, M. & Pestka, S., Methods in Enzymology, 79, 45 (1981).

Monoclonal antibodies were made against a synthetic polypeptide containing the last 16 amino acid residues of the C-terminal peptide of rIFN-γ. One of the monoclonal antibodies (Mo γ 2-11.1) was used for the purification of rIFN-γ. More specifically, the monoclonal antibodies and antibody affinity column of this invention were prepared as described in co-pending patent application PCT/JP83/00174, filed 5/31/83 in Japan and as detailed below in the following examples.

EXAMPLE 1

Synthesis of carrier protein-polypeptide complex used as antigen

The polypeptide H-Lys-Arg-Lys-Arg-Ser-Gln-Met-Leu-Phe-Arg-Gly-Arg-Arg-Ala-Ser-Gln-OH was coupled with thyroglobulin (hereinafter, TG) according to the method of Goodfriend et al. (Science, 144, 1334, 1964). The above-mentioned peptide can be produced by the conventional methods of peptide synthesis. Either of the solid phase method and liquid phase method may be used, although the liquid phase synthetic method is advantageous in many cases. Such methods of peptide synthesis are described, for example, by Schroder and Lubke in "The peptides",. vol. 1, Academic Press, New York, U.S.A., 1966, or by Izumiya et al. in "Peptide Syntheses", Maruzen, Tokyo, Japan, 1975, or by Haruaki Yajima in "Experiments in Biochemistry, vol. 1, pages 207-400", Tokyo Kagaku Dojin, 1977, and include, among others, the azide method, chloride method, acid anhydride method, mixed acid anhydride method, DCC method, active ester method, method using Woodward reagent K, carbodiimidazole method, oxidation reduction method and DCC/additive (e.g. HONB, HOBt, HOSu) method.

Said peptide can be produced by condensing a reactive carboxyl-containing starting material corresponding to one of the two fragments from division of said peptide at any site of the peptide bonding thereof with a reactive amino-containing starting material corresponding to the other fragment by any of the conventional peptide synthesis methods and, in case the condensation product has a protective group, eliminating the protective group in the conventional manner.

The method of protecting a functional group which should not be involved in the reaction between the materials, the protective group to be used in such protection, the method of eliminating such protective group and the method of activating the functional group to be involved in the reaction, for instance, can be selected adequately from among known ones or means.

Thus, 2.5 mg of said polypeptide was mixed with 3.75 mg of TG and, following addition of 2 ml of 50 mM phosphate buffer, the mixture was stirred well in ice water. Thereto was gradually added drop by drop a solution of 30.4 mg of carbodiimide hydrochloride in 200 ml of distilled water. Thereafter, the mixture was stirred in ice water for 3 hours. After the reaction, dialysis was performed against distilled water to a sufficient extent, followed by lyophilization to give 4.7 mg of a protein complex.

EXAMPLE 2

Preparation of antigen for Enzyme Immunoassay (EIA) for antibody detection

The antigen for EIA was prepared according to Kitagawa et al. (Journal of Biochemistry, vol. 79, page 233, 1976).

(i) Introduction of a maleimido group into the polypeptide

The polypeptide (350 nmoles) as obtained in Example 1 (xvi) was dissolved in 1 ml of 100 mM phosphate buffer (pH 6.8), and the solution was added to a solution of 585 ug (1.75 umoles) of N-(4-carboxycyclohexylmethyl)maleimide N-hydroxy-succinimide ester in 70 μl of N,N-dimethylformamide. The mixture was stirred at 30° C. for 30 minutes. After the reaction, fractionation was performed using a Sephadex G-25 column to give 185 nmoles of a polypeptide fraction with the maleimido group introduced therein.

(ii) Coupling of the maleimido-group-containing polypeptide with Beta-D-galactosidase

The maleimido-containing polypeptide (16.5 nmoles) as obtained in Example 3 (i) was mixed with 3.3 nmoles of Beta-D-galactosidase. After 18 hours of reaction at 4° C., 412,5 nmoles of Beta-mercaptoethanol was added for terminating the reaction. The Beta-D-galactosidase-coupled polypeptide was fractionated on a Sepharose 6B column and used for the subsequent experiments.

EXAMPLE 3

Immunization

Each of 6 female BALB/C mice aged 7 to 8 weeks was subcutaneously inoculated with 40 μg (on the protein basis) of the protein complex obtained in Example 1 (as the antigen) in intimate admixture with Freund's complete adjuvant (primary immunization). Two weeks after the primary immunization, the mice were subcutaneously inoculated with the antigen at the same dose as above in intimate admixture with Freund's incomplete adjuvant (secondary immunization). Further two weeks later, a third immunization was made in the same manner as in the secondary immunization. Six days after the third immunization, partial blood sampling was made from the mice and the serum antibody titers were determined by the EIA method described in Example 4. The mouse numbered γ-2 gave the highest antibody titer and was subjected to the final immunization by intravenous inoculation with 120 μg of the antigen dissolved in 0.5 ml of aqueous sodium chloride. The antibody titer data for each mouse are shown in Table 1.

                  TABLE 1                                                          ______________________________________                                         Antipeptide antibody titers in immunized mice                                          B/T (%)                                                                          Primary immu-                                                                              Secondary   Third immu-                                  Mouse No. nization.sup.(1)                                                                           immunization.sup.(2)                                                                       nization.sup.(3)                             ______________________________________                                         γ-1   --.sup.(4)                                                                               N.D         24.5                                         2           N.D.sup.(5)                                                                              19.3        35.3                                         3         --          N.D         24.7                                         4         N.D         1.3         1.7                                          5         N.D         1.8         5.0                                          6         --          N.D         0.8                                          Normal mouse                                                                             0.6         0.1         N.D                                          ______________________________________                                          .sup.(1) Serum dilution ratio: 1/1000                                          .sup.(2) Serum dilution ratio: 1/6300                                          .sup.(3) Serum dilution ratio: 1/7800                                          .sup.(4) --: Not detectable                                                    .sup.(5) ND: Not determined                                                    B/T: (Bound enzyme activity/total added enzyme activity) × 100     

EXAMPLE 4

Cell fusion

Immunization was performed by the method described in Example 5. Three days after the final immunization, the spleen was excised from the γ-2 mouse, filtered under pressure through a stainless mesh, and suspended in Eagle's minimum essential medium (MEM) to give a spleen cell suspension. For cell fusion, BALB/C mouse-derived P3-x63.Ag8.U1 (P3U1) myeloma cells were used (Current Topics in Microbiology and Immunology, vol. 81, page 1, 1978). Cell fusion was performed by the original method (Nature, vol. 256, page 495, 1975). Thus, spleen cells and P3U1 cells were separately washed three times with serum-free MEM and mixed at a ration of 5:1 (in number of cells). The mixture was centrifuged at 800 rpm for 15 minutes, whereby the cells were settled. After thorough removal of the supernatant, the sediment was lightly loosened, 0.3 ml of 45% polyethylene glycol (PEG) 6000⁻ (Koch-Light) was added, and the mixture was allowed to stand in a warm water tank maintained at 37° C. for 7 minutes so as to effect cell fusion. Thereafter, MEM was added thereto at a rate of 2 ml per minute. After addition of 12 ml in total of MEM, the resulting mixture was centrifuged at 600 rpm for 15 minutes, followed by removal of the supernatant. The cell sediment was suspended in RPMI-1640 medium supplemented with 10% fetal calf serum (RPMI1640-10FCS) in a concentration of 2×10⁵ P3U1 cells/ml and each of 144 wells on 24-well multidishes (Linbro) was seeded with 1 ml of the suspension. After seeding, the cells were incubated at 37° C. in a 5% carbon dioxide gas incubator. After 24 hours, HAT-selective culture was started by adding RPMI1640-10FCS medium supplemented with HAT (1×10⁻⁴ M hypoxanthine, 4×10⁻⁷ M aminopterin, 1.6×10⁻⁵ M thymidine) (HAT medium) in an amount of 1 ml per well. The HAT-selective culture was continued while 1 ml of the old medium was replaced by 1 ml of HAT medium 3,5 and 7 days after start of the culture. The growth of hybridomas was noted 10 to 14 days after cell fusion. When the culture broth turned yellow (about 1×10⁶ cells/ml), the supernatant was collected and examined for the presence of antibody by the EIA method. In this manner, supernatants from 141 wells in which hybridoma growth had been noted were examined. Two wells (γ2-11 and γ2-100) afforded intense antibody activity and other two wells (γ2-62 and γ2-70) presented weak antibody activity.

EXAMPLE 5

Cloning

Hybridomas from 3 wells (γ2-11, 62 and 100) which were positive in antibody activity were cloned by the limiting dilution method. Thus, hybridoma cells were suspended in RPMI1640-20FCS in a concentration of at least about 2 hybridoma cells/ml and the suspension was distributed on 0.1-ml portions into the wells on a 96-well microplate (Nunc). In said distribution, 5×10⁵ per well of BALB/C mouse thymocytes were added as feeder cells. As a result, cell proliferation was observed in about 2 weeks. The supernatant was then collected and examined for the present of antibodies by the EIA method as described in Example 4. Antibody activity was noted in 8 out of 19 clones from γ2-11 well, in 3 out of 54 clones from γ2-62 well, and in 5 out of 47 clones from γ2-100 well (Table 2).

                  TABLE 2                                                          ______________________________________                                         Anti-peptide antibody activity                                                 of cloned hybridomas                                                           Hybridoma No.   B/T (%)                                                        ______________________________________                                         γ2-11                                                                     1              68                                                              2              31                                                              3              63                                                              6              68                                                              7              67                                                              9              69                                                             12              42                                                             18              60                                                             γ2-62                                                                    14              20                                                             16              21                                                             34              16                                                             γ2-100                                                                    2              69                                                              3              70                                                             16              56                                                             25              80                                                             46              33                                                             Hyperimmune     35                                                             mouse serum                                                                    ______________________________________                                    

EXAMPLE 6

Binding capacity of monoclonal antibody to IFN-γ

The binding capacity of monoclonal antibody to IFN-γ was determined by the following method. To 300 ul of a 3% solution of cellulose coupled with rabbit anti-mouse IgG antibody, 300 ul of the culture supernatant for each of 2 or 3 cloned cell lines from each of γ2-11, γ2-62 and γ2-100 wells was added, and the reaction was allowed to proceed at room temperature for 12 to 20 hours. Thereafter, the cellulose was thoroughly washed with physiological saline, and 550 U/ml of IFN-γ obtained by the procedure mentioned below was added thereto. After 3 to 4 hours of reaction, the supernatant was collected and the IFN-γ obtained by the procedure mentioned below was added thereto. After 3 to 4 hours of reaction, the supernatant was collected and the IFN-γ activity therein was determined by the cytopathic effect (CPE) reading method using a microplate (Applied Microbiology, vol. 16, page 1706, 1968). Thus, 50 μ l of MEM was placed in each well of a 96-well microplate (Nunc) and 50 μl of a WISH cell suspension (4×10⁵ cells/ml) in 20% FCS-containing MEM was added, and incubation was conducted in a carbon dioxide gas incubator at 37° C. About 35 hours later, when cells in the IFN sample-free well showed 100% CPE, each well was microscopically observed for the estimation of CPE, and the reciprocal of the dilution factor for the IFN sample in that well in which 50% CPE was noted was referred to as the IFN titer.

The IFN-γ sample used was the supernatant collected 72 hours after stimulation of human peripheral lymphocytes with 40 μg/ml of concanavalin A and 15 ng/ml of 12-0-tetra-decanoylphorbol-13-acetate. Each ml of this culture supernatant contained 4400 units of human IFN-γ (acid-liabile and unstable to pH treatment). If antibodies having binding capacity to IFN-γ are present in the cloned cell culture supernatant, then the added IFN-γ should be conjugated to the antibodies on cellulose and reduction in IFN-γ activity of the supernatant should occur. As a result, for the clone γ2-11, relatively intense binding activity to IFN-γ was noted and 50-75% of the added IFN-γ (550 U/ml) was conjugated to antibodies (Table 3).

                  TABLE 3                                                          ______________________________________                                         Absorption of IFN-γ activity                                             by monoclonal antibodies                                                                      Remaining IFN activity                                                         (μ/ml)                                                       Hybridoma culture                                                                               Experiment                                                                               Experiment                                          supernatant      1         2                                                   ______________________________________                                         γ2-11.1    138       275                                                 γ2-11.2    207       N.D.                                                γ2-11.6    N.D.      275                                                 γ2-62.2    275       550                                                 γ2-62.3    275       550                                                 γ2-100.2   550       N.D.                                                γ2-100.3   550       N.D.                                                --               550       550                                                 ______________________________________                                    

EXAMPLE 7

Ascites formation by monoclonal antibody-producing hybridomas

Ascites formation was caused by intraperitoneal inoculation of BALB/C mice intraperitoneally pretreated with 0.5 ml of mineral oil with 1×10⁶, γ2-11.1 clone cells capable of producing antibodies having IFN-γ-binding activity. Ten days after intraperitoneal administration of hybridomas, the ascitic fluid was taken and examined for antibody activity, which was detected until 10⁷ -fold dilution. While the antibody activity of the corresponding clone cell culture supernatant was detected until 10⁴ -fold dilution, the formation of ascites (ascitization) led to an about 1000 times increase in antibody activity.

EXAMPLE 8

Monoclonal antibody purification

Using 4 ml of the ascitic fluid obtained in Example 9 as the starting material, monoclonal antibody purification was performed by the method of Staehelin et al. (Journal of Biological Chemistry, vol. 256, page 9750, 1981). Thus, the ascitic fluid was first centrifuged at 10,000 rpm for 15 minutes to remove fibrin-like substances therefrom and then diluted with phosphate buffer-saline (PBS: 8.1 mM NaH₂ PO₄, 1.5 mM KH₂ PO₄, 2.7 mM KCl, 137 mM NaCl; pH 7.2) to a concentration at which the ultraviolet absorption at 280 nm (A₂₈₀) for said dilution would range from 12 to 14. Thereafter, saturated aqueous ammonium sulfate was added to the sample dilution to a concentration of 47% for the sulfate. The mixture was stirred at 4° C. for 60 minutes to effect salting out and then centrifuged (10,000 rpm, 15 minutes) to give a precipitate. The precipitate was dissolved in 20 mM Tris buffer (pH 7.9) containing 50 mM NaCl and dialyzed against 2 liters of the same buffer. Two hours later, the dialyzing solution was replaced by a fresh 2-liter portion of the same solution and the dialysis was continued for further 15 hours. Thereafter, the precipitate was removed by centrifugation at 10,000 rpm for 15 minutes, and the supernatant was adjusted to a concentration such that the A₂₈₀ value became 20-30. This sample was subjected to fractionation on a DEAE-cellulose column (8 ml, Whatman, DE₅₂) equilibrated with a sufficient amount of Tris buffer containing 50 mM NaCl, elution being made with Tris buffer containing 50 mM NaCl at a flow rate of 1.5 ml/minutes. Under these conditions, the antibody activity was detected mainly in effluent fractions (FIG. 1). Antibody confirmation was made by SDS-poly-acrylamide gel electrophoresos (SDS-PAGE) method as described by Laemmli et al. (Nature, vol. 227, page 680, 1970). Thus, some of the fractions obtained by ammonium sulfate salting out and DEAE-cellulose fractionation were each subjected to reduction with 2-mercaptoethanol, followed by 17% SDS gel electrophoresis at 30 volts for 24 hours. In agreement with the antibody activity peaks, two bands were noted at positions corresponding to molecular weights of about 55 kilodaltons (H chain) and about 28 kilodaltons (L chain) (FIG. 2). The thus-purified antibody fraction 17 was examined for IFN-γ-binding activity by adding IFNγ (2200 U/ml). It was thus found that about 50% of IFN-γ was bound to the antibody (Table 4).

                  TABLE 4                                                          ______________________________________                                                                 Residual IFN activity                                  Sample         Dilution (μ/ml)                                              ______________________________________                                         γ2-11.1 fraction 17                                                                     10.sup.-1                                                                               1100                                                                  10.sup.-2                                                                               1100                                                                  10.sup.-3                                                                               2200                                                                  10.sup.-4                                                                               2200                                                   Anti-IgE monoclonal                                                                           10.sup.-1                                                                               2200                                                   antibody       10.sup.-2                                                                               2200                                                                  10.sup.-3                                                                               2200                                                                  10.sup.-4                                                                               2200                                                   ______________________________________                                    

EXAMPLE 9

Subclass to which monoclonal antibodies belong

The fraction 17 purified by the method of Example 8 was diluted 10 times and subjected to agar precipitation reaction (Ouchterlony test: Immunological Methods, Gel-Diffusion Technique, Blackwell, Oxford, 1964) using goat anti-mouse IgG1, G2a, G2b and G3 antibodies (Miles) so the IgG subclass to which γ2-11.1 monoclonal antibodies might belong could be identified. A single distinct band was found between the monoclonal antibody and the goat anti-mouse IgG2b antibody, while no band formation was noted between the monoclonal antibody and other anti-antibodies. Accordingly, said monoclonal antibody was found to belong to IgG2b (Table 5).

                  TABLE 5                                                          ______________________________________                                         Monoclonal antibody subclass                                                                                Precipitation                                     Antigen            Antibody  curve                                             ______________________________________                                         Monoclonal antibody of the                                                                        Anti-IgGl -                                                 present invention (fraction                                                    17)                                                                            Monoclonal antibody of the                                                                        Anti-IgG2a                                                                               -                                                 present invention (fraction                                                    17)                                                                            Monoclonal antibody of the                                                                        Anti-IgG2b                                                                               +                                                 present invention (fraction                                                    17)                                                                            Monoclonal antibody of the                                                                        Anti-IgG3 -                                                 present invention (fraction                                                    17)                                                                            ______________________________________                                    

EXAMPLE 10

Twenty-five ml (65.3 mg) of the monoclonal antibody from the effluent fractions as purified by the procedure of Example 8 was dialyzed overnight against 0.1M NaHCO₃ (pH 8.3). Separately, 25 ml of AFFI-GEL 10 (Bio-Rad) was thoroughly washed with water using a glass filter, suspended in 0.1M NaHCO₃ (pH 8.3) and mixed with the above antibody. The mixture was stirred gently at 4° C. for 4 hours to effect the reaction, and then allowed to stand at 4° C. overnight. The AFFI-GEL 10 was washed well with 0.1M NaHCO₃ (pH 8.3) using a glass filter. To the gel was added 25 ml of a solution (pH 8.0) containing 0.1M ethanolamine and 0.15M NaCl. The mixture was shaken at 4° C. for an hour so as to block possibly remaining unreacted active groups. Then, the gel was washed well with PBS, and suspended in 25 .ml of 0.1% NaN.sub. 3 -containing PBS. The suspension was stored at 4° C. Based on the amount of the added antibody and the amount of the antibody in the recovered filtrate, it was found that the antibody was conjugated to the gel in a proportion of 2.35 mg/ml of gel. A column was packed with the reaction product obtained in this manner and used as an antibody column.

EXAMPLE 11

All purification steps were carried out at 4° C. Frozen cells (25 g) were suspended in three volumes (75 ml) of 7M guanidine-HCI (pH7). The mixture was stirred for 1 h and then centrifuged for 1 h at 30,000×g. The supernatant was diluted 10-fold with Dulbecco's phosphate buffered saline (PBS) or 0.15M sodium borate buffer (pH9.5) and then centrifuged for 30 min. at 30,000×g. Alternatively, frozen cells (25 g) were suspended in 1.5 volumes (37.5 ml) of 0.15M sodium borate buffer (pH9.5) and stirred for 1 h. The mixture was sonicated 5 times for 30 seconds and then centrifuged for 1 h at 30,000×g. The supernatants from either guanidine-HCl extraction or sonification were mixed for 1 h on a rotating shaker with 25 ml silica (NuGel-952AC, Separation Industries, Metuchen, NJ), prewashed with phosphate buffered saline. The mixture was poured onto an empty column and the column was washed with 20-30 column volumes of 1M NaCl. The column was then eluted with 0.5M tetramethylammonium chloride in 0.01M sodium borate buffer (pH8.0). Interferon activity was eluted in about 200 ml and separated into 4 pools. Each pool was loaded onto a monoclonal antibody (MO γ 2-11.1) affinity column (4 ml bed volume) equilibrated with phosphate buffered saline. After washing with 10 column volumes of phosphate buffered saline buffer, the column was eluted with either 1M guanidine-HCl or 50% ethylene glycol containing 1M NaCl and 20 mM sodium phosphate buffer (pH7.0). Interferon activity was eluted in the first 20 ml.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed as described by Laemmli (reference 9). Protein was determined by fluorescamine analysis with crystalline bovine serum albumin as the reference standard. Interferon activity was determined by the cytopathic effect inhibition assay with vesicular stomattis virus and human WISH cells as reported in reference 10. All interferon titers are expressed in reference units/ml calibrated against the reference standard of partially purified human immune interferon.

A summary of the extraction purification procedure is presented in Table 6. The overall recovery was 25-32% and the purification was about 100 to 769 fold with an average specific activity of about 1×10⁷ units/mg. Total yield of rIFN-γ was 3-4 times higher with guanidine extraction (Table 6). Sodium dodecyl sulfate-polyacrylamide gel electrophresis of the last stage of purification is shown in FIG. 1. The material purified from guanidine extraction showed a single band at about 18,000 daltons (18K rIFN-γ) whereas the sonification procedure yielded a major band at about 15,000 daltons (15K rIFN-γ) and a minor band at about 17,000 daltons (FIG. 1A). Dimers and oligomers of rIFn-γ were formed (FIG. 1B) on non-reducing gel.

The 18K rIFN-γ was homogeneous and the amino acid composition was consistent with that predicted from the DNA sequence (see Table 7). Amino acid composition of the 15K and 18K rIFN-γ are given in Table 7. Several hundred picomoles of reduced and carboxymethylated 15K and 18K protein underwent Edman degradation in an automatic gas-phase protein/peptide sequencer. The amino acid N-terminal sequences of first 32 residues and 25 residues of, respectively, 15K and 18K proteins were in accord with that predicted by the DNA sequence (FIG. 2). ¹⁴ C-Carboxymethylated cysteines were detected in the first and third cycles of sequence analyses. No N-terminal methionine was detected. N-terminal sequence analysis of the 18K and 15K rIFN-γ demonstrated that the sequence of the area of both proteins is identical to that predicted from the DNA sequence. The C-terminal peptides have also been characterized to determine whether any deletions or changes are present in this region. Amino acid analysis of carboxypeptidase A (CPA) digestion mixture indicated that serine and/or glutamine (C-terminal amino acids) were released from the 18K rIFN-γ, whereas the 15K rIFN-γ was not digested by CPA under the same condition. Since Ser-Gln was the C-terminal sequence of rIFN-γ the 18K species appeared to have the intact C-terminal residues predicted from the DNA sequence and the 15K species has a different C-terminal residue (Lys) which is not cleaved by CPA.

The C-terminal residues predicted from the DNA sequences were further confirmed by analyzing and sequencing the C-terminal peptides after CNBr treatment. C-terminal peptides were separated on the HPLC C-18 reverse-phase column (FIG. 3). A sharp peptide peak (peak II), eluted from the early part of the gradient, was obtained from the 15K rIFN-γ and this peptide was absent from the CNBr digestion mixture of the 18K rIFN-γ. Amino acid analysis of this peptide indicated that this peptide has no homoserine or homoserine lactone and therefore must be the C-terminal CNBr peptide of the 15K protein (Table 8). Based on amino acid analysis (Table 8), this peptide corresponded to the amino acid residues No. 121-131 (no arginine was detectable). The sequence of the 11 amino acids was confirmed by sequence analysis. In the case 18K rIFN-γ, a relatively broad peak was obtained in the early part of the elution. This peak was further separated into two peaks by a shallow gradient. The amino acid analyses indicated that the first peak is the CNBr C-terminal peptide of the 18K protein (Table 8) and the amino acid composition matches the amino acid residues No. 138-146. The sequence of the 9 amino acids was verified by sequence determination. The C-terminal amino acid on the 15K protein was determined to by lysine.

These results indicated that the 18K species was the intact rIFN-γ molecule, whereas the 15K species was a proteolytic product. The peptide bond between amino acid residues No. 131 and No. 132 (Lys-Arg) was cleaved on the 15K species.

EXAMPLE 12

The 15K fragment of Immune interferon was purified to homogeniety from the mixture containing other fragments, i.e. the 17K fragment, by the following additional procedures. A sonification supernatent such as obtained in Example 10 (protein 10.3 mg protein; specific activity, 0.9×10⁶ U/mg) which has been purified through the monoclonal antibody affinity column was further purified by SDS-Page gel electrophoresis (16 slab gels; acrylamide 12.5%; SDS, 0.1% thickness, 3-5 mm). One slot of the gel was first stained with 0.1% Coomassie Brilliant Blue R-250 then destained with 10% acetic acid in 15% methanol. The corresponding 15K gel bands of other slots on the gel were cut out, crushed and stirred in 0.1% SDS at 4° C. for 2 days. The solution was then filtered on filter paper. The filtrate was lyphilized and then combined with 6 volumes of ethanol. The resultant precipitate was dissolved in 0.1% SDS to yield a solution of homogeneous 15K monomer component (protein, 1.1 mg; specified activity, 0.3×10⁶ U/mg).

EXAMPLE 13

In accordance with this invention the aforementioned novel immune interferon can be used for the same purposes as the other known interferons, e.g. as a prophylaxis or means for treating viral or neoplastic disorders or as a treatment for immunosupressive conditions. It may be administered in pharmaceutically acceptable oral, injectable or topical composition and modes. Dosage and dose rate may parallel that currently being used in clinical applications of the known interferons, typically about 1-200×10⁶ units daily. These pharmaceutical compositions of the invention contain said immune interferon in association with a compatible pharmaceutically acceptable carrier material. Any conventional carrier material can be utilized. The carrier material can be an organic or inorganic inert carrier material suitable for enteral, percutaneous or parenteral administration. Suitable carriers include water, gelatin, gum arabic, lactose, starch, magnesium stearate, talc, vegetable oils, polyalkylene-glycols, petroleum jelly and the like. Furthermore, the pharmaceutical preparations may contain other pharmaceutically active agents. Additional additives such as flavoring agents, preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.

The pharmaceutical preparations can be made up in any conventional form including: (a) a solid form for oral administration such as tablets, capsules, pills, powders, granules, and the like; (b) a liquid form for oral administration such as solutions, syrups, suspensions, elixirs and the like; (c) preparations for parenteral administration such as sterile solutions, suspensions or emulsions; and (d) preparations for topical administrations such as solutions, suspensions, ointments, creams, gels, micronized powders, aerosols and the like. The pharmaceutical preparations may be sterilized and/or may contain adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers, salts for varying the osmotic pressure and/or buffers.

Parenteral dosage forms may be infusions or injectable solutions which can be injected intravenously or intramuscularly. These preparations can also contain other medicinally active substances. Additional additives such as preservatives, stabilizers, emulsifying agents, buffers and the like may be added in accordance with accepted practices of pharmaceutical compounding.

                  TABLE 6                                                          ______________________________________                                         Purification of rIFN-γ                                                              Total   Total    Specific                                                                              Purifi-                                                Protein Activity Activity                                                                              cation-                                                                              Yield                                 Purification Step                                                                         mg      units    unit/mg                                                                               fold  %                                     ______________________________________                                         I.  Guanidine                                                                      extraction                                                                     Supernatant                                                                               2,806   2.5 × 10.sup.8                                                                  9 × 10.sup.4                                                                    --    100                                     Silica       98    1.0 × 10.sup.8                                                                  1 × 10.sup.6                                                                     11   40                                      Monoclonal    8    0.8 × 10.sup.8                                                                  1 × 10.sup.7                                                                    110   32                                      Antibody                                                                   II. Sonification                                                                   Supernatant                                                                               6,136   8.0 × 10.sup.7                                                                  1.3 × 10.sup.4                                                                  --    100                                     Silica       87    4.5 × 10.sup.7                                                                  5.2 × 10.sup.6                                                                  400   56                                      Monoclonal    2    2.0 × 10.sup.7                                                                  1.0 × 10.sup.7                                                                  769   25                                      Antibody                                                                   ______________________________________                                          Details of purification procedure were described under "Experimental           Procedures".                                                             

                  TABLE 7                                                          ______________________________________                                         Amino Acid Compositions of 15K and 18K rIFN-γ                            15K             18K     18K          15K                                       1-146               1-131                                                      (predicted value)                                                                              %146    (predicted value)                                                                           %131                                      ______________________________________                                         Asp  D     (20)         20.9  (20)       19.9                                  Thr  T     (5)          5.1   (5)        4.9                                   Ser  S     (9)          9.8   (11)       6.7                                   Glu  E     (16)         18.5  (18)       15.1                                  Pro  P     (2)          (2)*  (2)        (2)                                   Gly  G     (4)          5.9   (5)        5.6                                   Ala  A     (7)          8.2   (8)        7.3                                   Cys  C     (2)          (2)*  (2)        (2)                                   Val  V     (8)          9.1   (8)        9.1                                   Met  M     (3)          4.7   (4)        3.8                                   Ile  I     (7)          7.2   (7)        6.6                                   Leu  L     (9)          10.5  (10)       9.6                                   Tyr  Y     (5)          5.5   (5)        4.8                                   Phe  F     (9)          10.3  (10)       8.2                                   His  H     (2)          1.8   (2)        2.5                                   Lys  K     (19)         19.9  (20)       16.9                                  Arg  R     (3)          8.6   (8)        5.6                                   W          (1)          (1)*  (1)        (1)*                                  ______________________________________                                          ≠Figures in parenthesis indicate the predicted residue number from       DNA sequence.                                                                  *Values for Proline, cystine and tryptophan were not determined.         

                  TABLE 8                                                          ______________________________________                                         CNBr C-terminal Peptides of 15K and 18K                                                  15K              18K                                                 ______________________________________                                         Asp     D                                                                      Thr     T       0.9      .sup. (1).sup.≠                                 Ser     S       1.1      (1)     0.84   (1)                                    Glu     E       1.1      (1)     1.1    (1)                                    Pro     P       *        (1)                                                   Gly     G       1.5      (1)     1.3    (1)                                    Ala     A       2.4      (3)     1.1    (1)                                    Cys     C                                                                      Val     V                                                                      Met     M                                                                      Ile     I                                                                      Leu     L       1.0      (1)     1.1    (1)                                    Tyr     Y                                                                      Phe     F       1.0      (1)                                                   His     H                                                                      Lys     K       1.9      (2)     2.8    (3)                                    Arg     R                                                                      Positions   121-131            138-146                                         in                                                                             sequence                                                                       ______________________________________                                          Figures in parenthesis indicate the predicted residue number from DNA          sequence.                                                                      *Valve for Proline was not determined.                                   

REFERENCES

1. Gray, P. W. et al. (1982) Nature 295, 503-508.

2. Staehelin, T. et al. (1981) J. Biol. Chem. 256, 9750-9754.

3. Allen, G. Sequencing of Protein and Peptides. (1981) North-Holland Publishing Co., Amsterdam, New York pp. 30-31.

4. Amber, R. P. (1967) Methods in Enzymol. 11, 436-445.

5. Wolfe, R. A. and Stein, S. (1982) Modern Methods in Pharmacology pp. 55-77, Alan R. Liss, Inc. New York, NY.

6. Stein, S. and Brink, L. (1981) Methods in Enzymology, 79, 20-25.

7. Hewick, R. M. Hunkapillar, M. W., Hodd, L. E. and Dreyer, W. I. (1981) J. Biol. Chem. 256, 7990-7997.

8. Hawke, D., Yuan, P-M., and Shively, J. E. (1982) Anal. Biochem. 120, 302-311.

9. Laemmli, U. K. (1970) Nature 227, 680-685.

10. Rubinstein, S., Familletti, P. C., and Pestka, S. (1981) J. Virol. 37, 755-758.

11. Capon, D. J., Leung, D. W., Hitzeman, R. A., Perry, L. J., Kohr, W. J., Gray, P. W., Derynck, R., and Goddel, D. V. (1982) The Third Annual International Congress for Interferon Research, Miami, Fla. 

What is claimed is:
 1. A gamma interferon polypeptide fragment species with the amino acid sequence of the formula:

    ______________________________________                                         Cys  Tyr    Cys Gln  Asp Pro                                                                               Tyr Val                                                                               Lys Glu                                                                               Ala Glu                              Asn  Leu    Lys Lys  Tyr Phe                                                                               Asn Ala                                                                               Gly His                                                                               Ser Asp                              Val  Ala    Asp Asn  Gly Thr                                                                               Leu Phe                                                                               Leu Gly                                                                               Ile Leu                              Lys  Asn    Trp Lys  Glu Glu                                                                               Ser Asp                                                                               Arg Lys                                                                               Ile Met                              Gln  Ser    Gln Ile  Val Ser                                                                               Phe Tyr                                                                               Phe Lys                                                                               Leu Phe                              Lys  Asn    Phe Lys  Asp Asp                                                                               Gln Ser                                                                               Ile Gln                                                                               Lys Ser                              Val  Glu    Thr Ile  Lys Glu                                                                               Asp Met                                                                               Asn Val                                                                               Lys Phe                              Phe  Asn    Ser Asn  Lys Lys                                                                               Lys Arg                                                                               Asp Asp                                                                               Phe Glu                              Lys  Leu    Thr Asn  Tyr Ser                                                                               Val Thr                                                                               Asp Leu                                                                               Asn Val                              Gln  Arg    Lys Ala  Ile His                                                                               Glu Leu                                                                               Ile Gln                                                                               Val Met                              Ala  Glu    Leu Ser  Pro Ala                                                                               Ala Lys                                                                               Thr Gly                                                                               Lys                                  ______________________________________                                    

wherein the polypeptide has substantially the same activity as intact gamma interferon.
 2. The polypeptide of claim 1 as a homogeneous peptide.
 3. A gamma interferon polypeptide fragment species with the amino acid sequence of the formula:

    __________________________________________________________________________     Met                                                                               Cys                                                                               Tyr                                                                               Cys                                                                               Gln                                                                               Asp                                                                               Pro                                                                               Tyr                                                                               Val                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Glu                                           Asn                                                                               Leu                                                                               Lys                                                                               Lys                                                                               Tyr                                                                               Phe                                                                               Asn                                                                               Ala                                                                               Gly                                                                               His                                                                               Ser                                                                               Asp                                           Val                                                                               Ala                                                                               Asp                                                                               Asn                                                                               Gly                                                                               Thr                                                                               Leu                                                                               Phe                                                                               Leu                                                                               Gly                                                                               Ile                                                                               Leu                                           Lys                                                                               Asn                                                                               Trp                                                                               Lys                                                                               Glu                                                                               Glu                                                                               Ser                                                                               Asp                                                                               Arg                                                                               Lys                                                                               Ile                                                                               Met                                           Gln                                                                               Ser                                                                               Gln                                                                               Ile                                                                               Val                                                                               Ser                                                                               Phe                                                                               Tyr                                                                               Phe                                                                               Lys                                                                               Leu                                                                               Phe                                           Lys                                                                               Asn                                                                               Phe                                                                               Lys                                                                               Asp                                                                               Asp                                                                               Gln                                                                               Ser                                                                               Ile                                                                               Gln                                                                               Lys                                                                               Ser                                           Val                                                                               Glu                                                                               Thr                                                                               Ile                                                                               Lys                                                                               Glu                                                                               Asp                                                                               Met                                                                               Asn                                                                               Val                                                                               Lys                                                                               Phe                                           Phe                                                                               Asn                                                                               Ser                                                                               Asn                                                                               Lys                                                                               Lys                                                                               Lys                                                                               Arg                                                                               Asp                                                                               Asp                                                                               Phe                                                                               Glu                                           Lys                                                                               Leu                                                                               Thr                                                                               Asn                                                                               Tyr                                                                               Ser                                                                               Val                                                                               Thr                                                                               Asp                                                                               Leu                                                                               Asn                                                                               Val                                           Gln                                                                               Arg                                                                               Lys                                                                               Ala                                                                               Ile                                                                               His                                                                               Glu                                                                               Leu                                                                               Ile                                                                               Gln                                                                               Val                                                                               Met                                           Ala                                                                               Glu                                                                               Leu                                                                               Ser                                                                               Pro                                                                               Ala                                                                               Ala                                                                               Lys                                                                               Thr                                                                               Gly                                                                               Lys                                           __________________________________________________________________________

wherein the polypeptide has substantially the same activity as intact gamma interferon.
 4. A composition useful in the prophylaxis or treatment of viral, neoplastic or immunosuppressive disorders comprising a therapeutically inert pharmaceuticlly acceptable carrier and a therapeutically effective amount of a polypeptide with the amino acid sequence of the formula:

    ______________________________________                                         Cys  Tyr    Cys Gln  Asp Pro                                                                               Tyr Val                                                                               Lys Glu                                                                               Ala Glu                              Asn  Leu    Lys Lys  Tyr Phe                                                                               Asn Ala                                                                               Gly His                                                                               Ser Asp                              Val  Ala    Asp Asn  Gly Thr                                                                               Leu Phe                                                                               Leu Gly                                                                               Ile Leu                              Lys  Asn    Trp Lys  Glu Glu                                                                               Ser Asp                                                                               Arg Lys                                                                               Ile Met                              Gln  Ser    Gln Ile  Val Ser                                                                               Phe Tyr                                                                               Phe Lys                                                                               Leu Phe                              Lys  Asn    Phe Lys  Asp Asp                                                                               Gln Ser                                                                               Ile Gln                                                                               Lys Ser                              Val  Glu    Thr Ile  Lys Glu                                                                               Asp Met                                                                               Asn Val                                                                               Lys Phe                              Phe  Asn    Ser Asn  Lys Lys                                                                               Lys Arg                                                                               Asp Asp                                                                               Phe Glu                              Lys  Leu    Thr Asn  Tyr Ser                                                                               Val Thr                                                                               Asp Leu                                                                               Asn Val                              Gln  Arg    Lys Ala  Ile His                                                                               Glu Leu                                                                               Ile Gln                                                                               Val Met                              Ala  Glu    Leu Ser  Pro Ala                                                                               Ala Lys                                                                               Thr Gly                                                                               Lys                                  ______________________________________                                     